Botulinum neurotoxin (BoNT) is among the most potent toxins known and no therapy exists to terminate its action after intoxication. Because of the risk for BoNT use by bioterrorists, therapeutics for this agent are urgently needed. We propose developing parallel high-throughput screening (HTS) assays for BoNT drug discovery, based on cleavage of natural targets by this protease, a strategy that can be generalized to other class A biothreats such as anthrax lethal factor. Our approach utilizes in vitro HTS to allow rapid selection of drug candidates, along with a companion cell-based screens using cultured cells that report toxin activity. Importantly, two (2) versions of the cell-based system may identify therapeutics that interrupt different stages of the BoNT intoxication cycle while eliminating compounds that are generally cytotoxic, or cell-impermeable. The in vitro HTS is based on molecular engineering of the biological targets for different BoNT light chain (LC) protease serotypes, such as SNAP25 and VAMP. These substrates are modified to create fusion proteins with a reporter domain (eg., an enzyme or flourescent protein) on one (1) end and an immobilization domain (eg., 6x His) on the other. Cleavage by BoNT releases the reporter activity, which is then separated from non-cleaved target bound via the immobilization domain. For one (1) type of cell-based HTS stable BoNT sensitive cell lines which express the substrate-reporter proteins described above are intoxicated with LC. using one (1) of several methods (protein transduction of LC, viral vector delivery, transfection with inducible vector), after which cells are lysed and reporter is measured. For screening of activity in vivo, we propose two (2) strategies, both of which allow addition of holotoxin to a stable cell line engineered to report BoNT activity. In one (1), substrate protein, fused to a transcription factor domain, activates transcription of a suitable reporter enzyme upon cleavage by BoNT. In the other, real-time changes in bioluminescence resonance energy transfer (BRET) of a dual label substrate - reporter is measured after exposing cells to BoNT LC.